View source for UDP-glucose 4-epimerase

{{Short description|Class of enzymes}}
{{Redirect|GALE|the DARPA program|Global Autonomous Language Exploitation}}
{{infobox_gene}}
{{infobox enzyme
| Name       = UDP-glucose 4-epimerase
| EC_number  = 5.1.3.2
| CAS_number = 9032-89-7
| GO_code    = 0003978
| image      = Human GALE bound to NADH and UDP-glucose.png
| width      =
| caption    = ''H. sapiens'' UDP-glucose 4-epimerase homodimer bound to <span style="color:Goldenrod;">NADH</span> and <span style="color:magenta;">UDP-glucose</span>. Domains: <span style="color:blue;">N-terminal</span> and <span style="color:green;">C-terminal</span>. 
}}
{{infobox protein
|Name=UDP-galactose-4-epimerase
|caption=Human GALE bound to <span style="color:blue;">NAD+</span> and <span style="color:Goldenrod;">UDP-GlcNAc</span>, with <span style="color:green;">N-</span> and <span style="color:cyan;">C-terminal</span> domains highlighted. <span style="color:magenta;">Asn 207</span> contorts to accommodate UDP-GlcNAc within the active site.
|image=Human GALE bound to NAD+ and UDP-GlcNAc.png
|width=
|HGNCid=4116
|Symbol=GALE
|AltSymbols=
|EntrezGene=2582
|OMIM=606953
|RefSeq=NM_000403
|UniProt=Q14376
|PDB=
|ECnumber=5.1.3.2
|Chromosome=1
|Arm=p
|Band=36
|LocusSupplementaryData=-p35
}}
{{Pfam_box 
| Symbol =
| Name = NAD-dependent epimerase/dehydratase
| image = 
| width = 
| caption = 
| Pfam= PF01370
| InterPro= IPR001509
| SMART= 
| Prosite =          
| SCOP =     
| TCDB =  
| OPM family= 
| OPM protein= 
| PDB= 
|Membranome superfamily =330
}}

The [[enzyme]] '''UDP-glucose 4-epimerase''' ({{EnzExplorer|5.1.3.2}}), also known as '''UDP-galactose 4-epimerase''' or '''GALE''', is a homodimeric epimerase found in bacterial, fungal, plant, and mammalian cells. This enzyme performs the final step in the [[Leloir pathway]] of [[galactose]] metabolism, catalyzing the reversible conversion of [[UDP-galactose]] to [[UDP-glucose]].<ref name="Leloir">{{cite journal |vauthors=Holden HM, Rayment I, Thoden JB | title = Structure and function of enzymes of the Leloir pathway for galactose metabolism | journal = J. Biol. Chem. | volume = 278 | issue = 45 | pages = 43885–8 |date=November 2003 | pmid = 12923184 | doi = 10.1074/jbc.R300025200 | doi-access = free }}</ref> GALE tightly binds [[nicotinamide adenine dinucleotide]] (NAD+), a co-factor required for catalytic activity.<ref name="Liu_1996">{{cite journal |vauthors=Liu Y, Vanhooke JL, Frey PA | title = UDP-galactose 4-epimerase: NAD+ content and a charge-transfer band associated with the substrate-induced conformational transition | journal = Biochemistry | volume = 35 | issue = 23 | pages = 7615–20 |date=June 1996 | pmid = 8652544 | doi = 10.1021/bi960102v }}</ref>

Additionally, human and some bacterial GALE isoforms reversibly catalyze the formation of UDP-''N''-acetylgalactosamine (UDP-GalNAc) from UDP-''N''-acetylglucosamine ([[UDP-GlcNAc]]) in the presence of NAD+, an initial step in [[glycoprotein]] or [[glycolipid]] synthesis.<ref name="accommodate">{{cite journal |vauthors=Thoden JB, Wohlers TM, Fridovich-Keil JL, Holden HM | title = Human UDP-galactose 4-epimerase. Accommodation of UDP-N-acetylglucosamine within the active site | journal = J. Biol. Chem. | volume = 276 | issue = 18 | pages = 15131–6 |date=May 2001 | pmid = 11279032 | doi = 10.1074/jbc.M100220200 | doi-access = free }}</ref>

== Historical significance ==

Dr. [[Luis Leloir]] deduced the role of GALE in galactose metabolism during his tenure at the Instituto de Investigaciones Bioquímicas del Fundación Campomar, initially terming the enzyme waldenase.<ref name="pmid14885999">{{cite journal | author = LELOIR LF | title = The enzymatic transformation of uridine diphosphate glucose into a galactose derivative | journal = Arch Biochem | volume = 33 | issue = 2 | pages = 186–90 |date=September 1951 | pmid = 14885999 | doi = 10.1016/0003-9861(51)90096-3| hdl = 11336/140700 | hdl-access = free }}</ref> Dr. Leloir was awarded the 1970 [[Nobel Prize in Chemistry]] for his discovery of sugar nucleotides and their role in the biosynthesis of carbohydrates.<ref>{{cite press release | url = http://nobelprize.org/nobel_prizes/chemistry/laureates/1970/press.html | title=The Nobel Prize in Chemistry 1970 | publisher = The Royal Swedish Academy of Science | year = 1970 | access-date = 2010-05-17}}</ref>

== Structure ==

GALE belongs to the short-chain dehydrogenase/reductase (SDR) superfamily of proteins.<ref name="kavanagh">{{cite journal |vauthors=Kavanagh KL, Jörnvall H, Persson B, Oppermann U | title = Medium- and short-chain dehydrogenase/reductase gene and protein families : the SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes | journal = Cell. Mol. Life Sci. | volume = 65 | issue = 24 | pages = 3895–906 |date=December 2008 | pmid = 19011750 | pmc = 2792337 | doi = 10.1007/s00018-008-8588-y }}</ref> This family is characterized by a conserved Tyr-X-X-X-Lys motif necessary for enzymatic activity; one or more [[Rossmann fold]] scaffolds; and the ability to bind NAD<sup>+</sup>.<ref name="kavanagh"/>

=== Tertiary structure ===
GALE structure has been resolved for a number of species, including ''[[Escherichia coli|E. coli]]''<ref name="pmid10801319">{{PDB|1EK5}}; {{cite journal |vauthors=Thoden JB, Wohlers TM, Fridovich-Keil JL, Holden HM | title = Crystallographic evidence for Tyr 157 functioning as the active site base in human UDP-galactose 4-epimerase | journal = Biochemistry | volume = 39 | issue = 19 | pages = 5691–701 |date=May 2000 | pmid = 10801319 | doi = 10.1021/bi000215l}}</ref> and humans.<ref name="abortive">{{PDB|1XEL}}; {{cite journal |vauthors=Thoden JB, Frey PA, Holden HM | title = Molecular structure of the NADH/UDP-glucose abortive complex of UDP-galactose 4-epimerase from Escherichia coli: implications for the catalytic mechanism | journal = Biochemistry | volume = 35 | issue = 16 | pages = 5137–44 |date=April 1996 | pmid = 8611497 | doi = 10.1021/bi9601114 }}</ref> GALE exists as a homodimer in various species.<ref name="abortive"/>

While subunit size varies from 68 amino acids [https://www.ncbi.nlm.nih.gov/protein/ZP_05580029.1 (Enterococcus faecalis)] to 564 amino acids [https://www.ncbi.nlm.nih.gov/protein/YP_701629.1 (Rhodococcus jostii)], a majority of GALE subunits cluster near 330 amino acids in length.<ref name="kavanagh"/> Each subunit contains two distinct domains. An N-terminal domain contains a 7-stranded parallel β-pleated sheet flanked by α-helices.<ref name="Leloir"/> Paired [[Rossmann fold]]s within this domain allow GALE to tightly bind one NAD<sup>+</sup> cofactor per subunit.<ref name="Liu_1996"/> A 6-stranded β-sheet and 5 α-helices comprise GALE's C-terminal domain.<ref name="Leloir"/> C-terminal residues bind UDP, such that the subunit is responsible for correctly positioning UDP-glucose or UDP-galactose for catalysis.<ref name="Leloir"/>

===Active site===
The cleft between GALE's N- and C-terminal domains constitutes the enzyme's [[active site]]. A conserved Tyr-X-X-X Lys motif is necessary for GALE catalytic activity; in humans, this motif is represented by Tyr 157-Gly-Lys-Ser-Lys 161,<ref name="kavanagh"/> while ''E. coli'' GALE contains Tyr 149-Gly-Lys-Ser-Lys 153.<ref name="abortive"/> The size and shape of GALE's active site varies across species, allowing for variable GALE substrate specificity.<ref name="accommodate"/> Additionally, the conformation of the active site within a species-specific GALE is malleable; for instance, a bulky UDP-GlcNAc 2' N-acetyl group is accommodated within the human GALE active site by the rotation of the Asn 207 carboxamide side chain.<ref name="accommodate"/>

{| class="wikitable"
|+ Known ''E. coli'' GALE residue interactions with UDP-glucose and UDP-galactose.<ref name="dramatic">{{PDB|1A9Z}}; {{cite journal |vauthors=Thoden JB, Holden HM | title = Dramatic differences in the binding of UDP-galactose and UDP-glucose to UDP-galactose 4-epimerase from Escherichia coli | journal = Biochemistry | volume = 37 | issue = 33 | pages = 11469–77 |date=August 1998 | pmid = 9708982 | doi = 10.1021/bi9808969 }}</ref>
! width="200" | Residue
! width="600" | Function
|-
| Ala 216, Phe 218 || Anchor uracil ring to enzyme.
|-
| Asp 295 || Interacts with ribose 2' hydroxyl group.
|-
| Asn 179, Arg 231, Arg 292 || Interact with UDP phosphate groups.
|-
| Tyr 299, Asn 179 || Interact with galactose 2' hydroxyl or glucose 6' hydroxyl group; properly position sugar within active site.
|-
| Tyr 177, Phe 178 || Interact with galactose 3' hydroxyl or glucose 6' hydroxyl group; properly position sugar within active site.
|-
| Lys 153 || Lowers pKa of Tyr 149, allows for abstraction or donation of a hydrogen atom to or from the sugar 4' hydroxyl group.
|-
| Tyr 149 || Abstracts or donates a hydrogen atom to or from the sugar 4' hydroxyl group, catalyzing formation of 4-ketopyranose intermediate.
|}

== Mechanism ==

=== Conversion of UDP-galactose to UDP-glucose ===
GALE inverts the configuration of the 4' hydroxyl group of UDP-galactose through a series of 4 steps. Upon binding UDP-galactose, a conserved tyrosine residue in the active site abstracts a proton from the 4' hydroxyl group.<ref name="pmid10801319"/><ref name="tyr/ser">{{cite journal |vauthors=Liu Y, Thoden JB, Kim J, Berger E, Gulick AM, Ruzicka FJ, Holden HM, Frey PA | title = Mechanistic roles of tyrosine 149 and serine 124 in UDP-galactose 4-epimerase from Escherichia coli | journal = Biochemistry | volume = 36 | issue = 35 | pages = 10675–84 |date=September 1997 | pmid = 9271498 | doi = 10.1021/bi970430a }}</ref>

Concomitantly, the 4' hydride is added to the ''si''-face of NAD+, generating NADH and a 4-ketopyranose intermediate.<ref name="Leloir"/> The 4-ketopyranose intermediate rotates 180° about the pyrophosphoryl linkage between the glycosyl oxygen and β-phosphorus atom, presenting the opposite face of the ketopyranose intermediate to NADH.<ref name="tyr/ser"/> Hydride transfer from NADH to this opposite face inverts the stereochemistry of the 4' center. The conserved tyrosine residue then donates its proton, regenerating the 4' hydroxyl group.<ref name="Leloir"/>

=== Conversion of UDP-GlcNAc to UDP-GalNAc ===
Human and some bacterial GALE isoforms reversibly catalyze the conversion of UDP-GlcNAc to UDP-GalNAc through an identical mechanism, inverting the stereochemical configuration at the sugar's 4' hydroxyl group.<ref name="accommodate" /><ref name="pmid3948246">{{cite journal |vauthors=Kingsley DM, Kozarsky KF, Hobbie L, Krieger M | title = Reversible defects in O-linked glycosylation and LDL receptor expression in a UDP-Gal/UDP-GalNAc 4-epimerase deficient mutant | journal = Cell | volume = 44 | issue = 5 | pages = 749–59 |date=March 1986 | pmid = 3948246 | doi = 10.1016/0092-8674(86)90841-X| s2cid = 28293937 }}</ref>

== Biological function ==
[[File:Leloir pathway.png|thumb|right|300px|alt=Steps in the Leloir pathway of galactose metabolism.|Intermediates and enzymes in the Leloir pathway of galactose metabolism.<ref name="Leloir" />]]

===Galactose metabolism===
No direct catabolic pathways exist for galactose metabolism. Galactose is therefore preferentially converted into [[glucose-1-phosphate]], which may be shunted into [[glycolysis]] or the [[inositol]] synthesis pathway.<ref name="Lai_2009">{{cite journal |vauthors=Lai K, Elsas LJ, Wierenga KJ | title = Galactose toxicity in animals | journal = IUBMB Life | volume = 61 | issue = 11 | pages = 1063–74 |date=November 2009 | pmid = 19859980 | pmc = 2788023 | doi = 10.1002/iub.262 }}</ref>

GALE functions as one of four enzymes in the [[Leloir pathway]] of galactose conversion of glucose-1-phosphate. First, [[galactose mutarotase]] converts β-D-galactose to α-D-galactose.<ref name="Leloir" /> Galactokinase then phosphorylates α-D-galactose at the 1' hydroxyl group, yielding [[galactose-1-phosphate]].<ref name="Leloir" /> In the third step, [[galactose-1-phosphate uridyltransferase]] catalyzes the reversible transfer of a UMP moiety from UDP-glucose to galactose-1-phosphate, generating UDP-galactose and glucose-1-phosphate.<ref name="Leloir" /> In the final Leloir step, UDP-glucose is regenerated from UDP-galactose by GALE; UDP-glucose cycles back to the third step of the pathway.<ref name="Leloir" /> As such, GALE regenerates a substrate necessary for continued Leloir pathway cycling.

The glucose-1-phosphate generated in step 3 of the Leloir pathway may be isomerized to [[glucose-6-phosphate]] by [[phosphoglucomutase]]. Glucose-6-phosphate readily enters glycolysis, leading to the production of ATP and pyruvate.<ref name="isbn978-978-1-4292-3502-0">{{cite book | author1 = Stryer, Lubert | author2 = Berg, Jeremy Mark | author3 = Tymoczko, John L. | title = Biochemistry (Looseleaf) | publisher = W. H. Freeman | location = San Francisco | year = 2008 | pages = [https://archive.org/details/biochemistry3rdedi00stry/page/443 443–58] | isbn = 9780716718437 | url = https://archive.org/details/biochemistry3rdedi00stry/page/443 | url-access = registration }}</ref> Furthermore, glucose-6-phosphate may be converted to [[inositol-1-phosphate]] by [[inositol-3-phosphate synthase]], generating a precursor needed for [[inositol]] biosynthesis.<ref name="pmid18216771">{{cite journal | author = Michell RH | title = Inositol derivatives: evolution and functions | journal = Nat. Rev. Mol. Cell Biol. | volume = 9 | issue = 2 | pages = 151–61 |date=February 2008 | pmid = 18216771 | doi = 10.1038/nrm2334 | s2cid = 3245927 }}</ref>

=== UDP-GalNAc synthesis ===
Human and selected bacterial GALE isoforms bind UDP-GlcNAc, reversibly catalyzing its conversion to UDP-GalNAc. A family of [[glycosyltransferases]] known as UDP-''N''-acetylgalactosamine:polypeptide N-acetylgalactosamine transferases (ppGaNTases) transfers GalNAc from UDP-GalNAc to glycoprotein serine and threonine residues.<ref name="pmid12634319">{{cite journal |vauthors=Ten Hagen KG, Fritz TA, Tabak LA | title = All in the family: the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases | journal = Glycobiology | volume = 13 | issue = 1 | pages = 1R–16R |date=January 2003 | pmid = 12634319 | doi = 10.1093/glycob/cwg007 | doi-access = free }}</ref> ppGaNTase-mediated glycosylation regulates protein sorting,<ref name="pmid10359703">{{cite journal |vauthors=Alfalah M, Jacob R, Preuss U, Zimmer KP, Naim H, Naim HY | title = O-linked glycans mediate apical sorting of human intestinal sucrase-isomaltase through association with lipid rafts | journal = Curr. Biol. | volume = 9 | issue = 11 | pages = 593–6 |date=June 1999 | pmid = 10359703 | doi = 10.1016/S0960-9822(99)80263-2| s2cid = 16866875 | doi-access = free | bibcode = 1999CBio....9..593A }}</ref><ref name="pmid10712502">{{cite journal |vauthors=Altschuler Y, Kinlough CL, Poland PA, Bruns JB, Apodaca G, Weisz OA, Hughey RP | title = Clathrin-mediated endocytosis of MUC1 is modulated by its glycosylation state | journal = Mol. Biol. Cell | volume = 11 | issue = 3 | pages = 819–31 |date=March 2000 | pmid = 10712502 | pmc = 14813 | doi = 10.1091/mbc.11.3.819}}</ref><ref name="pmid11822873">{{cite journal |vauthors=Breuza L, Garcia M, Delgrossi MH, Le Bivic A | title = Role of the membrane-proximal O-glycosylation site in sorting of the human receptor for neurotrophins to the apical membrane of MDCK cells | journal = Exp. Cell Res. | volume = 273 | issue = 2 | pages = 178–86 |date=February 2002 | pmid = 11822873 | doi = 10.1006/excr.2001.5442 }}</ref><ref name="pmid10364244">{{cite journal |vauthors=Naim HY, Joberty G, Alfalah M, Jacob R | title = Temporal association of the N- and O-linked glycosylation events and their implication in the polarized sorting of intestinal brush border sucrase-isomaltase, aminopeptidase N, and dipeptidyl peptidase IV | journal = J. Biol. Chem. | volume = 274 | issue = 25 | pages = 17961–7 |date=June 1999 | pmid = 10364244 | doi = 10.1074/jbc.274.25.17961| doi-access = free }}</ref><ref name="pmid11878264">{{cite journal|author-link2=J. Evan Sadler |vauthors=Zheng X, Sadler JE | title = Mucin-like domain of enteropeptidase directs apical targeting in Madin-Darby canine kidney cells | journal = J. Biol. Chem. | volume = 277 | issue = 9 | pages = 6858–63 |date=March 2002 | pmid = 11878264 | doi = 10.1074/jbc.M109857200| doi-access = free }}</ref> ligand signaling,<ref name="pmid11287395">{{cite journal |vauthors=Hooper LV, Gordon JI | title = Glycans as legislators of host-microbial interactions: spanning the spectrum from symbiosis to pathogenicity | journal = Glycobiology | volume = 11 | issue = 2 | pages = 1R–10R |date=February 2001 | pmid = 11287395 | doi = 10.1093/glycob/11.2.1R| doi-access = free }}</ref><ref name="pmid11439191">{{cite journal |vauthors=Yeh JC, Hiraoka N, Petryniak B, Nakayama J, Ellies LG, Rabuka D, Hindsgaul O, Marth JD, Lowe JB, Fukuda M | title = Novel sulfated lymphocyte homing receptors and their control by a Core1 extension beta 1,3-N-acetylglucosaminyltransferase | journal = Cell | volume = 105 | issue = 7 | pages = 957–69 |date=June 2001 | pmid = 11439191 | doi = 10.1016/S0092-8674(01)00394-4| s2cid = 18674112 | doi-access = free }}</ref><ref name="pmid11081633">{{cite journal |vauthors=Somers WS, Tang J, Shaw GD, Camphausen RT | title = Insights into the molecular basis of leukocyte tethering and rolling revealed by structures of P- and E-selectin bound to SLe(X) and PSGL-1 | journal = Cell | volume = 103 | issue = 3 | pages = 467–79 |date=October 2000 | pmid = 11081633 | doi = 10.1016/S0092-8674(00)00138-0| s2cid = 12719907 | doi-access = free }}</ref> resistance to proteolytic attack,<ref name="pmid11150611">{{cite journal |vauthors=Sauer J, Sigurskjold BW, Christensen U, Frandsen TP, Mirgorodskaya E, Harrison M, Roepstorff P, Svensson B | title = Glucoamylase: structure/function relationships, and protein engineering | journal = Biochim. Biophys. Acta | volume = 1543 | issue = 2 | pages = 275–293 |date=December 2000 | pmid = 11150611 | doi = 10.1016/s0167-4838(00)00232-6}}</ref><ref name="pmid11294842">{{cite journal |vauthors=Garner B, Merry AH, Royle L, Harvey DJ, Rudd PM, Thillet J | title = Structural elucidation of the N- and O-glycans of human apolipoprotein(a): role of o-glycans in conferring protease resistance | journal = J. Biol. Chem. | volume = 276 | issue = 25 | pages = 22200–8 |date=June 2001 | pmid = 11294842 | doi = 10.1074/jbc.M102150200 | doi-access = free }}</ref> and represents the first committed step in mucin biosynthesis.<ref name="pmid12634319"/>

== Role in disease ==
{{main|Galactose epimerase deficiency}}
Human GALE deficiency or dysfunction results in Type III [[galactosemia]], which may exist in a mild (peripheral) or more severe (generalized) form.<ref name="Lai_2009"/>

== References ==
{{reflist|2}}

== Further reading ==
{{refbegin}}
* {{cite book | author = Leloir LF | chapter = Enzymic Isomerization and Related Processes | title = Advances in Enzymology and Related Areas of Molecular Biology | year = 1953 | journal = Adv. Enzymol. Relat. Subj. Biochem.  | volume = 14 | pages = 193–218  | pmid = 13057717 | doi = 10.1002/9780470122594.ch6 | series = Advances in Enzymology - and Related Areas of Molecular Biology | isbn = 9780470122594 }}
* {{cite journal |vauthors=Maxwell ES, de Robichon-Szulmajster H| year = 1960 | title = Purification of uridine diphosphate galactose-4-epimerase from yeast and the identification of protein-bound diphosphopyridine nucleotide | journal = J. Biol. Chem.  | volume = 235 | issue = 2 | pages = 308–312 | doi = 10.1016/S0021-9258(18)69520-1 | doi-access = free }}
* {{cite journal |vauthors=Wilson DB, Hogness DS| title = The enzymes of the galactose operon in Escherichia coli. I Purification and characterization of uridine diphosphogalactose 4-epimerase  | journal = J. Biol. Chem. | volume = 239 | pages = 2469–81 |date=August 1964 | doi = 10.1016/S0021-9258(18)93876-7 | pmid = 14235524 | doi-access = free }}
{{refend}}

==External links==
* [https://www.ncbi.nlm.nih.gov/books/NBK51671/  GeneReviews/NCBI/NIH/UW entry on Epimerase Deficiency Galactosemia]
* [https://www.ncbi.nlm.nih.gov/omim/230350,606953,230350,606953  OMIM entries on Epimerase Deficiency Galactosemia]  
* {{MeSH name|UDPgalactose+4-Epimerase}}

{{Fructose and galactose metabolism enzymes}}
{{Racemases and epimerases}}
{{Enzymes}}
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[[Category:EC 5.1.3]]
[[Category:Enzymes of known structure]]
[[Category:NADH-dependent enzymes]]